Rat Vascular Smooth Muscle Research Articles

Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

Diminished Rbfox1 increases vascular constriction by dynamically regulating alternative splicing of CaV1.2 calcium channel in hypertension.

Calcium influx from depolarized CaV1.2 calcium channels triggers the contraction of vascular smooth muscle cells (VSMCs), which is important for maintaining vascular myogenic tone and blood pressure. The function of CaV1.2 channel can be subtly modulated by alternative splicing (AS), and its aberrant splicing involves in the pathogenesis of multiple cardiovascular diseases. The RNA-binding protein Rbfox1 is reported to regulate the AS events of CaV1.2 channel in the neuronal development, but its potential roles in vascular CaV1.2 channels and vasoconstriction remain undefined. Here, we detect Rbfox1 is expressed in rat vascular smooth muscles. Moreover, the protein level of Rbfox1 is dramatically decreased in the hypertensive small arteries from spontaneously hypertensive rats in comparison with normotensive ones from Wistar-Kyoto rats. In VSMCs, Rbfox1 could dynamically regulate the AS of CaV1.2 exons 9* and 33. By whole-cell patch clamp, we identify knockdown of Rbfox1 induces the hyperpolarization of CaV1.2 current-voltage relationship curve in VSMCs. Furthermore, siRNA-mediated knockdown of Rbfox1 increases the K+-induced constriction of rat mesenteric arteries. In summary, our results indicate Rbfox1 modulates vascular constriction by dynamically regulating CaV1.2 alternative exons 9* and 33. Therefore, our work elucidates the underlying mechanisms for CaV1.2 channels regulation and provides a potential therapeutic target for hypertension.

PRMT5 critically mediates TMAO-induced inflammatory response in vascular smooth muscle cells

A high plasma level of the choline-derived metabolite trimethylamine N-oxide (TMAO) is closely related to the development of cardiovascular disease. However, the underlying mechanism remains unclear. In the present study, we demonstrated that a positive correlation of protein arginine methyltransferase 5 (PRMT5) expression and TMAO-induced vascular inflammation, with upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in primary rat and human vascular smooth muscle cells (VSMC) in vitro. Knockdown of PRMT5 suppressed VCAM-1 expression and the adhesion of primary bone marrow-derived macrophages to TMAO-stimulated VSMC. VSMC-specific PRMT5 knockout inhibited vascular inflammation with decreased expression of VCAM-1 in mice. We further identified that PRMT5 promoted VCAM-1 expression via symmetrical demethylation of Nuclear factor-κB p65 on arginine 30 (R30). Finally, we found that TMAO markedly induced the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) and production of reactive oxygen species, which contributed to PRMT5 expression and subsequent VCAM-1 expression. Collectively, our data provide novel evidence to establish a Nox4-PRMT5-VCAM-1 in mediating TMAO-induced VSMC inflammation. PRMT5 may be a potential target for the treatment of TMAO-induced vascular diseases.

MicroRNA-126 regulates the expression of inflammationrelated genes in vascular smooth muscle cells of diabetic rats

Purpose: To investigate the regulatory role of miR-126 in inflammation-associated gene expression in vascular smooth muscle (VSMCs) cells of diabetic rats.Methods: Diabetes was induced in rats by intraperitoneal injection of streptozocin (STZ) at a dose of 50 mg/kg. VSMCs were isolated from the aortic intimal-medial layers of the rats by a standard protocol. Expression of miR-126 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Transfection was performed with Lipofectamine 2000 reagent.Results: Expression of miR-126 was significantly (p < 0.05) downregulated in the VSMCs of diabetic rats. Similarly, expressions of SOD, APX and CAT were downregulated, while those of COX, LOX and NOS were significantly upregulated in VSMCs. However, transfection-induced miR-126 overexpression in the VSMCs of diabetic rats led to significant (p < 0.05) upregulation of SOD, CAT, APX and downregulation of COX, LOX and NOS. TargetScan analysis revealed that miR-126 exerted these effects by targeting SIRT1 gene. Furthermore, qRT-PCR and western blotting revealed that miR-126 overexpression in diabetic VMSCs caused significant (p < 0.05) upregulation of the expression of SIRT-1.Conclusion: The results indicate that miR-126 regulates the expression of inflammation-related genes by targeting SIRT-1 genes in vascular smooth muscle cells of diabetic rats. Thus, miR-126 may be beneficial in the management of diabetes.Keywords: Diabetes, Inflammation, Genes, microRNA, vascular smooth muscle cells

CONTRIBUTION OF ENVIRONMENTAL POLLUTANTS TO EPIGENETIC MODIFICATION IN RAT VASCULAR SMOOTH MUSCLE CELLS

Objective: More than a quarter of human diseases have been linked to exposure to environmental pollutants. These pollutants can exacerbate disease conditions through labile epigenetic modifications and directly result in changes in gene transcription. The most commonly reported epigenetic changes are acetylation and methylation of histone lysine. Contribution of acrolein, an environmental pollutant and a major component of cigarette smoke, to cardiovascular diseases and several neurological disorders has been reported. We have previously reported toxicity of acrolein in rat's vascular smooth muscle cells (VSMCs) and the effect of precursor of glutathione transferase, N-acetyl cysteine (NAC), in prevention of acrolein toxicity. In the present study, modification of histones and their effect on protein expression was further investigated in rat's VSMCs in the present/absent of acrolein and NAC. Design and method: VSMCs were treated with 3 μg/ml of acrolein for 6 and 24 hours in the present/absent of 0.2 mM NAC. At the end of the treatment, MTS essay, ELISA, western blot analysis, immunofluorescence, and LC/MS/MS analysis were used to check cells viability, superoxide dismutase (SOD) activity/expression, reactive oxygen species (ROS) generation, expression and cellular localization of H3K9 tri-methylation and acetylation, and identification of proteins affected by these changes. Results: Acrolein treated VSMCs exhibited the highest toxicity after 6 hours. Acrolein increased generation of ROS, reduced SOD activity and expression. There was 52% induction of acetylation and 62% in tri-methylation. Addition of N-acetyl cysteine reduced ROS, induced SOD (45%), reduced H3K9 acetylation by 102% and tri-methylation by 120%. These changes were confirmed with immunofluorescence staining. LC/MS/MS analysis revealed increased in actin protein and a significant decreased in annexin, heat shock cognate, and myosin 9 proteins. Conclusions: Based on our data we are concluding that effect of acrolein on VSCMs is partially due to alterations of H3K9 methylation/acetylation, resulting in overexpression of actin and significant reduction of annexin, heat shock cognate, and myosin 9 proteins. Addition of 0.2 mM NAC resulted in prevention of toxicity by acrolein by reducing ROS, improving SOD and preventing changes in H3K9 acetylation and methylation.

The intervention of enalapril maleate and folic acid tablet on the expressions of the GRP78 and CHOP and vascular remodeling in the vascular smooth muscle cells of H-hypertensive rats with homocysteine.

GRP78 and CHOP play essential roles in endoplasmic reticulum stress (ERS) of the vascular smooth muscle cells. We aim to investigate the effect of enalapril maleate and folic acid tablet on the expressions of GRP78 and CHOP and vascular remodeling in a homocysteine (HCY)-treated hypertensive rat model. The hypertensive rat model was established with the technique of coarctation in the abdominal aorta, and the blood pressure of the rat was measured with the non-destructive tail-cuff method two weeks after operation. Thirty-six rats with hypertension were randomly divided into 3 groups (n=12 in each group). The control group received common diet and double distilled water, methionine group received 30 g/L methionine diet and double distilled water, while enalapril maleate and folic acid tablet group received 30 g/L methionine diet and 0.2 mg.kg-1.d-1 solution of enalapril maleate and folic acid tablet. Samples were collected at week 4 and week 8 for analysis. The plasma homocysteine was measured by homocysteine detector; MAP was detected through carotid artery incubation and aortic media thickness was determined by image analyses software. The expression of GRP78 and CHOP in the vascular smooth muscle cells were identified by immunohistochemistry and Western blot. Compared with the control group, the concentration of HCY in the serum of rats in methionine group was increased significantly after 4 weeks (p < 0.01), and even more significant after 8 weeks (p < 0.01). Compared with that of methionine group, the level of HCY in enalapril maleate and folic acid tablet group rats was significantly decreased (p < 0.01). The level of MAP in methionine group was increased significantly after 8 weeks compared with that of control group (p < 0.05). However, the MAP in enalapril maleate and folic acid tablet group was decreased significantly compared with that of methionine group. Compared with control group, the media thickness of vascular smooth muscle of rats in the methionine group was increased significantly (p < 0.05) while was statistically reduced in the enalapril maleate and folic acid tablet group (p < 0.05). The expressions of GRP78 and CHOP in methionine group were significantly elevated compared to that of control in a time dependent manner (p < 0.05), which were remarkably down regulated in enalapril maleate and folic acid tablet group compared with that in methionine group. The administration of enalapril maleate and folic acid tablet can maintain the normal state of cells via the alleviation of ERS and vascular damages, reduction of HCY and the thickness of arterial media as well as the improvement of vascular remodeling.

Inhibitory effects of solid lipid nanoparticles of rhynchophylline on proliferation of rat vasculars mooth muscle cells induced by TGF-β1

Objective To observe the effect of Rhy-SLN on the proliferation of rat vascular smooth muscle cells (VSMC) induced by TGF-β1, and explore the mechanism. Methods The primary culture of rat thoracic aortic vascular smooth muscle cells was studied by tissue block culture method. The cells were divided into the control group, TGF-β1 group, TGF-β1+ the high, medium and low dosage groups of Rhy-SLN. In addition to the control group, the cells of the other groups were involved in the intervention of TGF-β1 of 20 g/L, and the high, medium and low dosage groups of Rhy-SLN cells were involved in the intervention of 25, 50, 100 mg/L of the hook teng solid lipid nanoparticles. After 24 hours of culture, MTT assay was used to detect cell proliferation inhibition rate in each group, and the cell cycle was detected by flow cytometry. The expression of c-myc and c-Fos protein in each group was detected by Western blot method. Results Compared with the TGF-β1 group, the absorbance value (0.457 ± 0.046 vs. 0.975 ± 0.049) of TGF-β1+ rhy-sln high dose group significantly decreased (P<0.01); the number of S phase cells (15.87% ± 2.47%, 15.23% ± 1.69%, 17.02% ± 2.87% vs. 38.58% ± 2.68%) of TGF-β1+ rhy-sln in each dose group significantly decreased (P<0.01); The c-myc (48.65 ± 3.65, 50.69 ± 4.16, 55.29 ± 3.67 vs. 68.21 ± 3.25) and c-Fos (38.78 ± 4.25, 43.56 ± 3.69, 46.58 ± 3.57 vs. 66.54 ± 4.09) of the TGF-β1+ rhy-sln each dose group significantly decreased (P<0.01). Conclusions The Rhy-SLN can inhibit the proliferation of VSMC in rats induced by TGF-β1.Its mechanism is related to the conversion of G0/G1 phase to the S phase and the expression of the reduction of c-myc and c-fos protein. Key words: Rhynchophylline; Solid lipid nanoparticles; Transforming growth factor beta; Proto-oncogene proteins c-fos; Proto-oncogene proteins c-myc; Muscle, smooth, vascular; Cell proliferation; Rat

Calcium/calmodulin-dependent kinase 2 mediates Epac-induced spontaneous transient outward currents in rat vascular smooth muscle.

Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain.In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown.In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.

Acrolein‐Induced Epigenetic Modification of Histone 3 in Rat Vascular Smooth Muscle Cells

ObjectiveEnvironmental pollutants can exacerbate disease conditions by affecting chromatin structure and ultimately regulating gene expression. Acetylation of histone lysine has been reported to contribute to gene expression while the methylation results in gene inactivation. Acrolein, an environmental pollutant and a major component of cigarette smoke, has been implicated in cardiovascular toxicity and several neurological disorders such as Alzheimer's disease. N‐acetyl cysteine (NAC), a precursor of glutathione transferase, has been reported to prevent acrolein toxicity in cell culture. The goal of the present study was to investigate the effects of acrolein on modification of histone 3, lysine 9 residue (H3K9) in rat's vascular smooth muscle cells (VSMCs) in the present and absent of NAC.Design and methodVSMCs were treated with 2 and 3 μg/ml of acrolein for 1, 6 and 24 hours in the present or absent of 0.2 mM NAC. At the end of the treatment, MTS essay was used to check cells viability. Western blot analysis was used to determine changes in H3K9 tri‐methylation and acetylation. Additionally, immunofluorescence staining was used to analysis cellular localization of H3K9 acetylation and methylation.ResultsCells treated with 2 and 3 μg/ml of acrolein exhibited the highest toxicity at 6 hours incubation. Acrolein exposed VSMC displayed induction of histone H3K9 acetylation for all concentrations and time periods. The most significant induction of acetylation (30% at 2 μg/ml] and 46% [3 μg/ml]) and tri‐methylation (37% [2 μg/ml] &amp; 65% [3 μg/ml]) occurred at 6 hours incubation. Co‐treated VSMCs with acrolein and N‐acetyl cysteine reduced H3K9 acetylation (100% for both 2 and 3 μg/ml), and tri‐methylation (115% &amp; 125% for 2 and 3 μg/ml respectively). Immunofluorescence staining further confirmed changes in acetylation and tri‐methylation in acrolein treated VSCMs with or without NAC.ConclusionsBased on our data we are concluding that effect of acrolein on VSCMs is partially due to alterations of H3K9 methylation and acetylation which can be prevented with addition of 0.2 mM NAC.Support or Funding InformationNIH SC3GM103746‐03

Evidence For Phosphorylation-Dependent Modulation of BK Channel Activity in Vascular Smooth Muscle (VSM) Myocytes and Resistance Arteries

VSM excitability is regulated by large conductance, calcium-activated K+ (BK) channels. We have previously reported that type I cGMP-dependent protein kinase (cGKI) phosphorylates the BK channel at key serine residues, leading to augmented BK current magnitude. Here, we investigate the cellular mechanisms regulating the temporal pattern (i.e. onset and decay) of stimulus-evoked, phosphorylation-dependent enhancement of BK current in VSM.BK currents were recorded from primary and cultured rat VSM myocytes by whole-cell patch clamp. Proximity ligation assay (PLA) was used to examine the co-localization of BK channels with key regulatory proteins (spatial restriction <40 nm). For pressure myography, isolated rat resistance arteries (∼200 micron diameter) were cannulated on glass pipettes and examined under constant intraluminal pressure (i.e. 70 mmHg).Voltage clamp recordings demonstrated that VSM BK current was reversibly enhanced by the nitrovasodilator sodium nitroprusside (SNP, 100 uM) and sensitive to the highly-selective BK channel inhibitor, penitrem A (100 nM). Calyculin A (30 nM), an inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), augmented both the magnitude and duration of the SNP-evoked BK current. Fostriecin (30 nM), a selective PP2A inhibitor, similarly increased BK current magnitude and duration. PLA fluorescence staining demonstrated the co-localization of BK channels with cGKI, which increased following SNP treatment. PLA staining also confirmed the co-localization of BK channels with PP2A protein. In myogenically-active arteries, SNP (10 uM) evoked a reversible inhibition of myogenic tone that was decreased by penitrem A. Fostriecin enhanced the magnitude and duration of the SNP effect on myogenic tone. Collectively, these data suggest that cGKI and PP2A form a macromolecular complex with BK channels to regulate the magnitude and duration of the NO-mediated enhancement of BK current in VSM.

Abstract MP08: Syndecan-1 is Involved in Osteoprotegerin-induced Vascular Dysfunction

Osteoprotegerin (OPG), an inhibitor of vascular calcification, has pleiotropic vascular effects independently of its actions on calcification. OPG has been associated with vascular inflammation and remodelling and may be important in cardiovascular disease where OPG levels may be elevated. Molecular mechanisms and functional consequences of OPG stimulation in the vasculature are unclear. We propose that syndecan-1, a membrane glycoprotein, may be important and that reactive oxygen species (ROS) play a role in OPG signalling. Vascular reactivity of resistance arteries from WKY rats was studied by wire myography in the presence or absence of OPG (50 ng/mL) and/or synstatin (SSNT - 10-6M - syndecan-1 inhibitor). Rat endothelial cells (EC) and vascular smooth muscle cells (VSMC) were studied. Levels of ROS were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine; peroxynitrite - ONOO-). Protein oxidation and levels were measured by immunoblotting. Exposure of resistance arteries to OPG induced endothelial (decreased relaxation to acetylcholine) and VSMC (decreased relaxation to sodium nitroprusside - SNP) dysfunction, as well as, increased contraction to phenylephrine. All responses were blocked by SSNT, N-acetylcisteine (antioxidant) and ML171 (Nox inhibitor). In EC, OPG-induced ROS production (240±46.1% increase vs. veh, p&lt;0.05) was blocked by SSNT. OPG decreased H2O2 production/release (61±5.4% vs. veh) and increased eNOS Thr 495 phosphorylation (inhibitory site) (100±24% vs. veh, p&lt;0.05). In VSMC, OPG increased H2O2 (69±3%) and ONOO- (43±12%) levels, protein oxidation (61±15%), Rho kinase (200±39%) and myosin light chain activation (55±3%) (all vs. veh, p&lt;0.05). Increase in OPG-induced ONOO- levels was exacerbated by SNP (130±16% vs. veh, p&lt;0.05), a nitric oxide donor. In conclusion, vascular dysfunction elicited by OPG is mediated by syndecan-1 and ROS. Whether syndecan-1 also impacts on OPG-sensitive calcification is unclear. Our data identify a novel molecular mechanism through syndecan- 1/ROS that may underlie injurious effects of OPG.

Polyunsaturated fatty acid biosynthesis is involved in phenylephrine-mediated calcium release in vascular smooth muscle cells

Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction.

Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells

Ethnopharmacological relevanceDodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. Material and methodsThe effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. Results100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. ConclusionThis study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase.

TRPV4 Channel Cooperativity in the Resistance Vasculature

TRPV4 Channel Cooperativity in the Resistance Vasculature

Epidermal Growth Factor Enhances Orthovanadate-Induced Contraction via Src and Myosin Phosphatase Target Subunit 1 in Rat Vascular Smooth Muscle

Inhibition of protein tyrosine phosphatase by orthovanadate induces vasoconstriction, which is mediated by the Rho kinase-dependent inactivation of myosin light chain phosphatase (MLCP) via signaling downstream of Src-induced activation of the epidermal growth factor (EGF) receptor. The present study investigated the potential role of EGF in orthovanadate (OVA)-dependent vaso-constriction. OVA-induced aortic contraction significantly increased in the presence of EGF, and was abolished by inhibitors of Rho kinase (Y27632), extracellular signal-regulated kinase 1 and 2 (Erk1/2) (FR180204), Erk1/2 kinase (PD98059), EGF receptor (AG1478), and Src (PP2). Treatment of the rat endothelium-denuded thoracic aorta with either EGF or OVA augmented the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr-853 and of the EGF receptor at Tyr-1173. The phosphorylation of MYPT1 was further increased by co-stimulation with EGF and OVA. EGF receptor phosphorylation at Tyr-845 was also increased by EGF or OVA; this effect was augmented by co-stimulation with EGF and OVA, and was abolished by Src inhibition. In addition, Erk1/2 was phosphorylated by EGF or by co-treatment with EGF and OVA; this was abolished by an EGF receptor inhibitor, but not by Src inhibition. These results suggested that OVA-induced EGF-related contraction was mediated by the Rho kinase-dependent inactivation of MLCP via two different signaling cascades: Src-dependent phosphorylation of the EGF receptor at Tyr-845 and EGF-dependent phosphorylation of Erk1/2.

Redox Control of the Senescence Regulator Interleukin-1α and the Secretory Phenotype

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H2O2 and intracellular Ca(2+) levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca(2+) promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca(2+) chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca(2+) chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca(2+) homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.

AB0075 Toll-like receptor 3 and interleukine 1b expression in circulating progenitor cells isolated from patients with rheumatoid arthritis.

BackgroundCirculating progenitor cells (CPCs), including CD34+ cells and endothelial progenitor cells (EPCs), play a role in delaying atherosclerosis and cardiovascular disease. Toll-like receptor 3 (TLR3), a member of the pathogen...

Identification and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

Impaired calcium mobilization in vascular smooth muscle of rats in septic shock

Impaired calcium mobilization in vascular smooth muscle of rats in septic shock

Angiotensin II regulates the LARG/RhoA/MYPT1 axis in rat vascular smooth muscle in vitro

To identify a key protein that binds monomeric G protein RhoA and activates the RhoA/Rho kinase/MYPT1 axis in vascular smooth muscle cells (VSMCs) upon angiotensin II (Ang II) stimulation. Primary cultured VSMCs from Sprague-Dawley rats were transfected with siRNAs against leukemia-associated RhoGEF (LARG), and then treated with Ang II, losartan, PD123319, or Val(5)-Ang II. The target mRNA and protein levels were determined using qPCR and Western blot analysis, respectively. Rat aortic rings were isolated, and the isometric contraction was measured with a force transducer and recorder. Stimulation with Ang II (0.1 μmol/L) for 0.5 h significantly increased the level of LARG mRNA in VSMCs. At 3, 6, and 9 h after the treatment with Ang II (0.1 μmol/L) plus AT(2) antagonist PD123319 (1 μmol/L) or with AT(1) agonist Val(5)-Ang II (1 μmol/L), the LARG protein, RhoA activity, and phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) in VSMCs were significantly increased. Knockdown of LARG with siRNA reduced these effects caused by AT(1) receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished. Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT(1), thereby maintaining vascular tone.